We have continued studies of the distribution of the histone variants H3.3 and H2A.Z in chicken erythroid cells. We had shown recently that H3.3 is concentrated at many regulatory regions, as well as over the transcribed regions of some active genes. We then showed that nucleosomes containing H3.3 are less stable than those containing the predominant species, H3.1, and lose their H2AH2B components more readily. Furthermore, nucleosomes containing H3.3 and H2A.Z are even more unstable. Using double chromatin immunoprecipitation and gentle isolation methods we have identified numerous genomic loci at where such doubly substituted nucleosomes are present. They mark transcriptional regulatory sites that control active genes. This work has now been extended to a genome-wide survey in a human cell line of the distribution of nucleosomes containing these histone variants, in collaboration with the laboratory of Dr. Keji Zhao, NHLBI. The data strongly confirm our earlier conclusions, and reveal strikingly different distribution patterns for single and double variant containing nucleosomes.